The copy number abnormality of chromosomes is closely linked with human diseases. The chromosome abnormality occurs in fetal cells carried with genetic disease and tumor cells. On average, 9 of 1000 newborns may carry diseases caused by copy number abnormality of chromosomes (1). Therefore, it is important to detect the copy number of chromosomes before children have not been born yet. However, currently used diagnosis methods including amniocentesis and ovine chorionic belong to invasive methods, which bring certain risks to pregnant women and fetuses. Serum protein markers and ultrasonic waves are used for detecting whether the fetuses suffer diseases due to copy number abnormality of chromosomes, although it is noninvasive, pathogenic factors are not detected directly, so the accuracy and sensitivity is not good (2). There is also a problem that the diseases due to the copy number abnormality of chromosomes cannot be found as soon as possible. This situation prompts researchers to develop an accurate and highly sensitive noninvasive diagnosis and detection method.
Since fetal DNA in maternal blood have been found (3), diagnosing and detecting the abnormality of fetal chromosomes noninvasively and directly becomes an important study topic. In 2007, Professor LO Yuk Ming Dennis and his colleagues proved that the percentage of mutation site of placenta specific gene 4 in maternal plasma mRNA could be used for judging whether the fetus has chromosome 21 which was a triploid (4). The percentage of mutation site is at the same time used for judging whether chromosome 18 is a triploid (5). Its limitation is in that the mutation site is not common in the crowd, so these methods are only suitable for a part of crowd. During the same period, digital PCR (dPCR) is used for detecting the triploid of fetal chromosome (6), (7). The digital PCR has the advantage of independency of any mutation site, but its accuracy is insufficient, and requires many blood samples, which increases sampling difficulty.
In recent years, the above problems have been solved by rapidly developed high-throughput DNA sequencing techniques. These techniques include Genome Analyzer of IIlumina (8), SOLiD of Life Technologies (9) and Heliscope of Helicos (10), by which hundreds of millions or even billions of sequences can be detected once. When these techniques are used for detecting DANs in maternal plasma, the change of the number of chromosomes of trace amounts of fetal DNA in plasma can be detected (11), (12), (13). But due to high sequencing cost, these techniques have not been used commonly. At the same time, there is still an unsolved problem of detecting the change of partial copy number of fetal chromosomes from maternal plasma. It is advantageous to detect the change of copy number of fetal chromosomes from maternal plasma by high-throughput sequencing, but this technique is expensive in cost and cannot be popularized. Moreover, the sequencing Coefficient Of Variation (CV) is high, and the detecting accuracy and stability also needs to be improved. The sequencing CV also decides that this method is only suitable for a few chromosomes, such as chromosome 21, chromosome 18, and unsuitable for detecting the change of partial copy number of chromosomes.
It is very expensive and difficult to detect the change of the number of chromosomes by high-throughput sequencing, the main reason is that the content of fetal DNA in maternal plasma is low, only 5% when it is low, in particular during early fetal development. Most DNA in maternal plasma is maternal DNA. The background of maternal DNA easily encompasses the change of the number of fetal chromosomes or partial copy number. Therefore, the method for separating the pregnant women and fetal DNA becomes the subject studied for years with little progress. A successful method should belong to histone separation method invented by Baylor Medical College (14). The quantity of DNA separated is very small, so the method is only suitable for detecting the mutation site, unsuitable for detecting the change of the copy number of chromosomes.